1. Field of the Invention
The present invention relates to structural analysis of nucleic acids, and more particularly to an apparatus for detecting denaturation of nucleic acids.
2. Related Background Art
Much attention has been attracted to techniques for detecting and measuring in denaturation of nucleic acids (DNA, RNA, and hybrid thereof) with accuracy which substantially resolves a difference in the denaturation conditions based on single base (or single base-pair) substitution, deletion or addition.
As a method capable of detecting slight differences such as the above-mentioned single base alteration between homologous double-strand nucleic acids, there has been proposed a method called "denaturing gradient gel electrophoresis method" (S. G. Fischer and L. S. Lerman: Proc. Natl. Acid. Sci. U.S.A., Vol. 80, pp. 1579-1583, March 1983). In this method, a double-strand nucleic acid to be measured is first amplified using an ordinary technique such as polymerase chain reaction (hereinafter, referred to as "PCR method"). Then, the resultant double-strand nucleic acid is charged into a gel carrier (or gel support), and the double-strand nucleic acid is subjected to electrophoresis under a gradient of a denaturation condition (such as temperature or hydrogen ion concentration (pH)) which is spatially provided across the gel carrier. In this measurement, it is possible to simultaneously subject a control double-strand nucleic acid functioning as a reference (or standard) together with the double-strand nucleic acid to be measured so as to observe a difference in the denaturation condition between these two species of the double-strand nucleic acids (i.e., the double-strand nucleic acid to be measured and the reference double-strand nucleic acid).
FIGS. 9A and 9B are schematic diagrams illustrating the above-mentioned conventional method for detecting the denaturation of a nucleic acid. For example, FIG. 9A relates to a case wherein temperature is selected as a denaturation condition, and schematically shows a state wherein a nucleic acid is denatured on the basis of temperature increase. In general, a double-strand nucleic acid is denatured when the ambient temperature is elevated to a predetermined value. The temperature at which a double-strand nucleic acid is denatured (denaturation temperature) depends on the composition of bases constituting the double-strand nucleic acid. A double-strand nucleic acid comprising a specific single-strand nucleic acid and another single-strand nucleic acid binding thereto which is completely complementary to the former single-strand nucleic acid, has a denaturation temperature higher than that of a double-strand nucleic acid comprising the above-mentioned specific single-strand nucleic acid and another single-strand nucleic acid binding thereto which is substantially (and not completely) complementary to the former single-strand nucleic acid.
Conventional apparatuses for detecting denaturation of a nucleic acid utilize such a phenomenon, and conduct gel electrophoresis while spatially providing a gradient of a denaturation condition in the gel carrier to be used for the electrophoresis.
FIG. 9B shows results of measurement relating to a case of a double-strand nucleic acid sample comprising a specific strand and another strand which is substantially complementary to the specific strand. As shown in FIG. 9B, when two denaturation points (i.e., points at which denaturation occurs) are present, one double-strand nucleic acid showing a denaturation condition of a lower temperature is the double-strand nucleic acid to be measured, and the other double-strand nucleic acid showing a higher temperature denaturation is the reference double-strand nucleic acid.
However, in the above-mentioned denaturation detection of a nucleic acid utilizing the conventional denaturing gradient gel electrophoresis method, it is necessary to subject the sample nucleic acid to electrophoresis in a gel across which a gradient or change in denaturation condition (temperature or pH) is provided. Accordingly, such a conventional denaturation detection method is troublesome or tedious. More specifically, the conventional method consumes a long period of time corresponding to several tens of minutes to several hours, while such a period of time depends on the molecular weight of the sample nucleic acid.
In addition, the sensitivity of detection of the nucleic acid denaturation by the conventional denaturing gradient gel electrophoresis method depends on the stability of the system to be used for such a purpose, the accuracy in the control of the denaturation condition such as degree of gradient to be provided to the gel, or the accuracy in the control of the condition for electrophoresis. However, it is difficult to improve the accuracy in the control of these conditions as compared with those accomplished at the present stage. Accordingly, there has been posed a problem such that further improvement is difficult with respect to the sensitivity and accuracy (or precision) of the denaturation detection.